Notwithstanding intensive research for a vaccine in the 20 years since the initial discovery of HIV as the causative agent of Acquired Immunodeficiency Syndrome (AIDS) and 18 years since the molecular cloning and characterization of the AIDS virus, major obstacles remain for HIV immunotherapy development. These hurdles include the hypervariability of HIV type 1 (HIV-1, the major HIV type), the multiple routes and/or modes of virus transmission, and a lack of understanding of the immune responses necessary for inhibition of HIV.
In a review by Moore et al. [1], it is reported that antibodies generated against variable exposed epitopes have effective neutralizing activity only against autologous viruses and isolates with closely-related envelope proteins (env). Further, the monoclonal antibodies (mAbs) that recognize more conserved regions of env, targets of better immunotherapeutic potential, almost never strongly neutralize multiple primary isolates. The basic knowledge needed for achievement of broad anti-viral antibodies remains so enigmatic that the field of HIV vaccinology is now focused on approaches which offer at best only a hope for partial protection against disease [2].
Primary isolates of HIV-1 are obtained by limited cultivation of patient peripheral blood mononuclear cells (PBMCs) or patient plasma with uninfected PBMCs. They closely resemble HIV field strains responsible for human infection [3]. Primary isolates are readily distinguished from commonly used laboratory-adapted T-tropic viruses such as IIIb/LAI, SF2, and MN, which have been reproduced and are well-adapted to grow in human T-lymphoid cell lines.
First, most primary HIV-1 isolates are M-tropic. They do not readily grow in cultured T cell lines, but rather they are monocyte or macrophage-tropic with the ability to infect primary T cells [4]. Second, primary isolates are highly resistant to in vitro neutralization by recombinant soluble forms of the viral receptor protein CD4 (rsCD4) and require 200-2700 times more rsCD4 than laboratory strains for comparable neutralization [5]. Third, primary isolates are also resistant to neutralizing antibodies elicited by the use of gp120 vaccines [6].
Primary isolates include both syncytium-inducing isolates (SI) and non-syncytium-inducing (NSI) isolates in PBMC culture. Most SI primary isolates will replicate in the highly HIV-sensitive T cell line MT2, but few can replicate in the less permissive transformed T cell lines CEM or H9 that are commonly used to culture laboratory-adapted isolates. Non-syncytium-inducing (NSI) primary isolates can only be cultured in the primary T cells from peripheral blood.
There was mistaken early optimism for efficacious antibody responses to recombinant HIV-1 envelope subunit vaccines (e.g., gp120 and gp160 vaccine products). The vaccinee sera from clinical trials of experimental envelope-based vaccines often were capable of neutralizing laboratory isolates of HIV-1 in vitro [7,8]. At the time, the basic differences between laboratory-adapted and primary isolates were not yet understood. This optimism was soon shaken when the antibodies in the vaccinee sera were found to be largely ineffective in neutralizing HIV-1 primary patient isolates [6,9].
Early optimism for effective vaccine-induced antibodies also was mistakenly brought about by studies of inactivated virus preparations of Simian Immunodeficiency Virus (SIV). Due to similarities between HIV-1 and SIV in morphology, genetic organization, infection and disease processes, SIV infection in rhesus monkeys seemed to be an excellent model to explore different AIDS vaccine and anti-HIV antibody strategies. Early studies in the SIV model showed that inactivated preparations of SIV grown on human T cell lines and formulated in adjuvant can protect macaques from infection after experimental inoculation with highly infectious, pathogenic variants of human cell-grown SIV [10]. Unexpectedly, this protection was lost when an SIV stock grown on homologous monkey cells was used for the challenge of immunized animals.
Later immunization studies with monkey cell-grown SIV and uninfected human cells showed that protection from infection in those early SIV studies probably resulted from the stimulation of immune responses to xenogeneic human host cell proteins rather than to virus-encoded antigens [11]. Passive immunization experiments involving SIV suggested that certain anti-cell antibodies may contribute to protection against SIV infection in the absence of cell-mediated immunity [12].
The mechanism by which anti-cell antibodies provides protective immunity to retroviral challenge has not been delineated. One mode of action for such protection may be mediated by blocking the CD4 binding site for immunodeficiency viruses on immune system cells. Anti-CD4 monoclonal antibodies have long been known to block infection in a manner that is dependent on the CD4 epitope, not the virus strain [13,14]. Monoclonal antibody 5A8, which recognizes CD4 domain 2, had shown therapeutic efficacy in SIV-infected macaques [15]. Anti-CD4 monoclonal antibodies Leu3A and P1, which recognize the CDR2-like loop in CD4 domain 1, blocked infection of cell cultures by primary isolates [5,14].
Anti-CD4 antibodies inhibit virus-to-cell or infected cell-to-uninfected cell transmission of both SI and NSI strains of HIV 1 [16]. The surface of host T cells appears to have a host cell antigen complex associated with CD4 that facilitates viral binding and entry and acts as a target for protective anti-cell antibodies [17,18].
Murine monoclonal antibody B4 (mAb B4)[17,18] is an anti-cell antibody that was developed by using CD4-expressing T lymphocytes, such as peripheral blood mononuclear T cells, thymocytes, splenocytes and leukemia or lymphoma-derived T cell lines, e.g., HPB-ALL or SUP-T1, as the immunogen. MAb B4 was characterized by its ability to neutralize primary isolates of HIV-1 and related immunodeficiency viruses both in vitro and in vivo. Specifically, mAb B4 acts by blocking the binding of HIV to CD4+ host cells. It is an entry inhibitor, a new class of anti-retroviral drugs for the treatment of HIV infection, providing potentially an orthogonal therapy to highly active antiretroviral therapy (HAART).
MAb B4 was directed against a host cell surface antigen complex comprising CD4 protein in association with domains from chemokine receptors. MAb B4 recognizes CD4 by a CD4 extracellular region binding site on the CDR2-like loop in domain 1. This is not coincident with the binding site for Leu3A. U.S. Pat. No. 5,912,176 discloses strong evidence that this B4 antibody has broad neutralizing activities against primary isolates from all clades of HIV-1 and primary isolates of HIV type 2 (HIV-2) and SIV. When monoclonal antibodies with specificities for various cell surface antigens were mixed, only those mixtures including mAb B4 displayed strong neutralizing activity for HIV-1 B-clade primary field isolates. (Column 24, lines 1-10; Column 47, lines 5-10.) Furthermore, in neutralization assays on primary isolates of HIV-2 (i.e., HIV-2ROD) and SIV strains, mAb B4 exhibited strong neutralization for all strains whereas no comparative result was shown by the anti-N-terminal V3 MN antibody previously shown to be effective on laboratory-adapted HIV-1 MN. (Column 45, lines 19-35.)
Additional studies of mAb B4 neutralizing activity for HIV-1 and primary isolates of HIV-2 and SIV demonstrate its efficacy in mice, chimpanzees, and rhesus macaques. For example, the administration of mAb B4 after infectious challenge, interrupted the infection by PBL-grown HIV-1 of hu-peripheral blood leukocyte (PBL)-severe combined immunodeficient mice [18]. Also, the administration of mAb B4 in chimpanzees after infectious challenge totally interrupted the infection by chimp-adapted HIV-1 [18]. Furthermore, following challenge with a dose of SIVmac251 that caused persistent infection in rhesus macaques, passive immunization of these animals with a modest dose (4 mg/kg) of mAb B4 is found to effectively protect 75 percent of the monkeys from infection by an SIV primary isolate, (U.S. Pat. No. 5,912,176, Column 50, lines 15-22.)
The requirement for CD4 as the receptor for efficient HIV infection suggests that the CD4 molecule may be a good target for immunotherapy by anti-cell antibodies like mAb B4, as long as there is not undue immunomodulatory effects.
However, the use of murine monoclonal antibodies like mAb B4 for therapeutic and in vivo diagnostic applications in man has been found to be limited by immune responses made by patients to the murine antibodies. The development of “HAMA” (human anti-murine antibody) responses in patients has limited the ability of murine antibodies to reach their antigenic targets and reduced the effectiveness of the antibodies in therapeutic use.
Antibody humanization technologies have been devised to reduce the HAMA response. For example, murine antibodies can be converted to chimeric mouse/human antibodies wherein the entire DNA coding sequences for the variable domains of the mouse immunoglobulin are joined to the regions encoding the human constant domains [19]. Mouse DNA sequences can be further reduced for better humanization by complementarity determining region (CDR) grafting. A rat Fv region was reshaped for use in human immunotherapy by excising the DNA coding regions for the six CDRs from the rat heavy and light chain variable regions and grafting them into the coding regions for the framework sequences of the human heavy and light chains. The reshaped variable region coding sequence was then assembled onto human constant domains [20].
However, chimeric and engrafted antibodies in which the variable region or CDRs remain murine may still be immunogenic. Moreover, grafting CDRs onto unrelated frameworks may lead to loss in affinity of the humanized antibody. Consequently, humanization of murine monoclonal antibodies was further applied by employing sequence homology and molecular modeling. These methods were used to select more homologous human frameworks for the murine CDRs that retain high-binding affinity and minimize the use of murine residues to those essential for contact [21].
Since the purpose for humanizing therapeutic murine antibodies is to reduce immunogenicity in human recipients, deimmunization is a useful alternative process. Deimmunization reduces the immunogenicity of rodent variable domains in humans simply by removing epitopes from the variable domains of rodent antibodies that are likely to be immunogenic in humans.
In practice, an effective primary immune response against a therapeutic antibody involves: the processing of foreign proteins, presentation of antigenic peptides by MHC class II molecules (T-cell epitopes), and then stimulation of helper T-cells. In turn, these helper T-cells trigger and enhance the B cell production of antibodies which bind to the therapeutic antibodies. Furthermore, if one or more sequences within a therapeutic antibody (B-cell epitopes) is bound by immature B-cell surface immunoglobulins (sIg) in the presence of suitable cytokines, the B-cell can be stimulated to differentiate and proliferate to provide soluble forms of the original sIg. These soluble forms of the original sIg can complex with the therapeutic antibody to limit its effectiveness and facilitate its clearance from the patient. Therefore, to avoid a primary immune response against the therapeutic mAb, both the B- and T-cell epitopes within the antibody that are potentially antigenic in humans should be eliminated or modified.
Without either the B or T-cell response, the primary immunogenic response to a therapeutic antibody is likely to be muted or absent. The DeImmunisation® technology developed by Biovation Ltd. (Aberdeen, UK) [22,23] focuses on the removal of potentially immunogenic B cell and T cell epitopes. This method was applied in the present invention to deimmunize mAb B4. Removal of B cell epitopes is achieved by the ‘veneering’ of surface residues with substituent amino acids [24]. Removal of the T cell epitopes is achieved following the identification of such epitopes from the variable regions of the therapeutic antibodies whereby the sequences of the variable region can be analyzed by the presence of MHC class II-binding motifs by a 3-dimensional “peptide threading” method [25]. The antibody constant regions of the murine antibody are replaced by the human antibody constant regions in the final deimmunized antibody by chimerization [19].
Passive immune therapy with human, humanized or deimmunized antibody may play an important role in the treatment or prevention of HIV infection. Human anti-HIV monoclonal antibodies 2F5 and 2G12 are known to neutralize primary isolates of HIV and have been used for studies in HIV-infected humans. Transient reduction in viral loads and transient increases in CD4+ T cells were observed [26]. These antibodies also have been studied in a non-human primate model for immune prophylaxis against mother-to-child transmission. Whether the antibodies were administered through the placenta by intravenous infusion into the mother prenatally or by direct infusion into rhesus macaque neonates following birth, the neonates were protected from challenge by infectious simian/human immunodeficiency virus (SHIV). The authors concluded that immunoprophylaxis with a combination of anti-viral monoclonal antibodies is a promising approach to prevent maternal HIV transmission in humans [27]. Passive immunity has also been proposed for use in post-exposure prophylaxis against HIV in the event of accidental exposure to HIV [16,18].
Since certain CD4-specific monoclonal antibodies can efficiently block infection of lymphocytes and macrophages by primary isolates of HIV-1 with a breadth higher than that of neutralizing anti-HIV antibodies [5,18,28], receptor-directed monoclonal antibodies like mAb B4 may be even better candidates for prophylaxis against HIV exposure and for therapy of HIV infection. Toward this purpose, monoclonal antibody 5A8 (mAb 5A8) has been previously reconfigured into a humanized IgG4 antibody by a CDR engraftment method [28]. As an IgG4 isotype, the humanized mAb 5A8 lacks a glycosylation site on the CH2 domain that is associated with complement fixation. This feature should improve the safety profile of similar antibodies by making it less likely to cause depletion of CD4+ lymphocytes in patients. Humanized 5A8 has been entered into a clinical trial in HIV-infected patients [29].
Additionally, anti-CD4 monoclonal antibodies have shown clinical benefit in humans with rheumatoid arthritis [30]. A human monoclonal antibody, HuMax-CD4, has been entered into clinical trial for treatment of rheumatoid arthritis and psoriasis [31]. These applications demonstrate the potential usefulness of deimmunized antibodies such as those derived from B4 antibodies as immunotherapeutic drugs against Human Immunodeficiency Virus (HIV) and CD4-mediated autoimmune disorders.